Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5-46 nm) are transported into and out of embryos through chorion pore canals (CPCs) and exhibit Brownian diffusion (not active transport), with the diffusion coefficient inside the chorionic space (3 x 10(-9) cm(2)/s) approximately 26 times lower than that in egg water (7.7 x 10(-8) cm(2)/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities.

We have synthesized and characterized stable (non-aggregation, non-photobleaching and nonblinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10 -11 to 1.3×10 -8 cm 2 /s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in-vivo assays to screen their biocompatibility.

We report here, for the first time, the use of a simple washing approach to reduce the ionic strength of the solution, which increased thickness of electric double layer on the surface of silver (Ag) nanoparticles, and thereby enhanced their surface zeta potential. This approach allowed us to prepare optically uniform (75-99%) and purified Ag nanoparticles (11.3 ± 2.3 nm) that are stable (non aggregation) in solution for months, permitting them to become robust and widely-used single nanoprobes for in vivo optical imaging. These Ag nanoparticles show remarkable photostability and serve as single nanoparticle photonic probes for continuous imaging nano-environments of segmentation-stage zebrafish embryos for hours. Unlike other particle tracking experiments, we utilized size-dependent localized-surface-plasmon-resonance-spectra (LSPRS) (colors) of single Ag nanoparticles to determine given colored (sized) nanoparticles in situ and used the monodisperse color (size) of nanoparticles to simultaneously measure viscosities and flow patterns of multiple proximal nano-environments in segmentation-stage zebrafish embryos in real-time. We found new interesting counterclockwise flow patterns with rates ranging from 0.06 to 1.8 μm/s and stunningly high viscosity gradients spanning two-orders of magnitude in chorion space of the embryos, with the highest viscosity observed around the center of chorion space and the lower viscosity at the interfacial areas near the surface of both chorion layers and inner mass of the embryos. This study demonstrates the possibility of using individual monodisperse nano-photonics to probe the roles of embryonic fluid dynamics in embryonic development.

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm).

Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding their structures and functions for design of effective therapies. In this study, we designed and synthesized two silver nanoparticle (Ag NP) solution with average diameters of 13.1 ± 2.5 nm (8.1–38.6 nm) and 91.0 ± 9.3 nm (56–120 nm), and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas (P.) aeruginosa in real-time at nanometer resolution. We found that the accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (over-expression of MexAB-OprM), but less than ΔABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton-motive-force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that the MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed longer inside the cells than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multi-sized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.